Methods and Results— Male LDL receptor−/− mice, that were either AT1a receptor +/+ or −/−, were fed a fat enriched diet and infused with either saline or AngII. AngII-induced augmentation of atherosclerosis and formation of AAAs was ablated in AT1a receptor−/− mice. Bone marrow transplantation studies were performed to determine the role of AT1a receptors expressed on infiltrating cells. AT1a receptor +/+ and −/− mice were irradiated and repopulated with bone marrow–derived stem cells of either genotype. These 4 groups of chimeric mice were infused with either saline or AngII. Repopulation of irradiated AT1a receptor +/+ mice with −/− bone marrow–derived cells resulted in modest reductions in AngII-induced atherosclerosis. Unexpectedly, AT1a receptor–deficient recipient mice were dramatically protected from AngII-induced vascular pathologies, irrespective of donor genotype.

Conclusion— AngII promotes vascular pathology via AT1a receptors. AT1a receptors expressed on infiltrating cells exert modest regulation of AngII-induced atherosclerosis. However, the presence of this receptor in resident tissue is required for the initiation of AngII-induced atherosclerosis and AAAs.

Angiotensin II (AngII) infusion into either LDL receptor−/− or apoE−/− mice leads to acceleration of atherogenesis and the development of abdominal aortic aneurysms (AAAs).1–4 AngII-induced atherosclerosis is characterized by intimal infiltration of leukocytes that become engorged with lipid. In contrast, AngII induction of AAAs is characterized by medial destruction, macrophage infiltration, thrombus formation, and vascular remodeling.5 Thus, AngII provides a common stimulus for atherosclerosis and AAAs, but the development of these pathologies appears to occur via distinct mechanisms.6 The specific receptor regulating the formation of AngII-induced atherosclerosis has not been determined. A role for AT1 receptors in AngII-induced AAAs has been demonstrated by inhibition with losartan, although the contribution of AT1a versus AT1b receptors has not been defined.7

Atherosclerosis and AAAs display distinct histological features during initiation and progression, but have the common feature of macrophage infiltration throughout the progression of both of these vascular diseases. AT1 receptors have been detected on macrophages.8,9 AngII promotes several potentially atherogenic effects on cultured macrophages, including enhanced expression of 12/15 lipoxygenase, production of peroxide, promotion of LDL oxidation, reduced cholesterol efflux, and augmented cholesterol synthesis.8,10–13 AT1 receptors are also present on resident cells of the arterial wall, including endothelium and smooth muscle cells. Incubation of AngII with cultured endothelial cells promotes the atherogenic mechanism of increased leukocyte adhesion that is associated with increased expression of several adhesion molecules, such as E-selectin and vascular cell adhesion molecule (VCAM)-1.14–16 AngII also stimulates several potential atherogenic mechanisms in smooth muscle cells, including secretion of MCP-1 and increased production of reactive oxygen species.17,18 In contrast, the AngII-induced mechanisms in these cell types that relate to the development of AAAs have not been as extensively characterized.

Because infiltration of leukocytes occurs in AngII-induced atherosclerotic lesions and AAAs, we hypothesized that AngII interacts with a specific receptor on infiltrating leukocytes to initiate the formation of vascular diseases. In initial studies, we defined the effects of AT1a receptor deficiency on the development of AngII-induced atherosclerosis and AAAs. After definition of the angiotensin receptor subtype mediating AngII-induced atherosclerosis and/or AAA formation, we used bone marrow transplantation to determine the role of the angiotensin receptor in donor versus recipient cells in the formation of these vascular pathologies. The transplantation protocol was validated to ensure that chimeric mice reproducibly expressed the donor phenotype in bone marrow–derived cells. Using this validated protocol, we performed these studies on mice in which both wild-type and receptor-deficient recipients were irradiated and repopulated with bone marrow–derived cells that were either receptor wild-type or deficient. Creation of these 4 groups of chimeric mice enabled distinction of the location of angiotensin receptor effects to the donor versus the recipient genotype.

To induce hypercholesterolemia, male mice (8 weeks of age) were fed a diet supplemented with saturated fat (milk fat 21%) and cholesterol (0.15%; Harlan Teklad; Diet #TD88137). One week after initiation of fat feeding, saline or AngII (1,000 ng/kg/min) was administered subcutaneously via Alzet osmotic minipumps (Model 2004) as described previously.2 All studies were performed with the approval of the University of Kentucky Institutional Animal Care and Use Committee.

Genotyping by Polymerase Chain Reaction

PCR screening for AT1a and LDL receptors was performed as described previously.19

Bone Marrow Transplantation

This procedure was performed as described previously.20–22 Mice were maintained on antibiotic water (sulfratrim, 4 μg/mL) for one week before irradiation. Recipient mice were irradiated with a total of 900 Rads from a cesium source that was delivered in two doses within 3 to 4 hours. Bone marrow–derived cells for CD45.1, AT1a receptor +/+ or −/− × LDL receptor−/− mice were obtained from the tibias and femurs of donor mice and were injected into the tail vein of 8-week-old irradiated recipient mice (1×107 cells per mouse). Mice were maintained on antibiotic water for 4 weeks after irradiation, then placed on regular water. For mice used in vascular pathology studies, six weeks after irradiation, the mice were fed a diet enriched in saturated fat and cholesterol (Teklad #TD88137) for 5 weeks. Osmotic mini-pumps containing drugs were implanted 1 week after the initiation of fat feeding.

Measurement of Serum Components

Serum cholesterol concentrations were determined by a commercially available enzymatic assay kit (Wako Chemicals). Lipoprotein cholesterol distribution was performed by size exclusion chromatography as described previously.2

Serum autoantibodies titers to modified lipoproteins were determined as described previously, with data expressed as the ratio of chromagen development for LDL compared with malondialdehyde-modified LDL coated plates.23

Blood Pressure Measurements

Systolic blood pressure was measured on conscious, restrained mice using the Visitech tail cuff system, as described previously.2

Quantification of Atherosclerosis and AAA

Atherosclerosis was quantified on the aortic arch as described previously.23,24 The maximum width of the abdominal aorta was measured using computerized morphometry (Image-Pro). Aneurysm incidence was quantified based on a definition of an external width of the suprarenal aorta that was increased by 50% or greater compared with aortas from saline-infused mice.25

Tissue Composition

Aortic tissues were sectioned and histologically stained using Movat pentachrome and immunostained for macrophages as described previously.26

Statistics

Data were analyzed with two way ANOVA using SigmaStat. Data were tested for use of parametric or nonparametric post hoc analysis, and multiple comparisons were performed using Tukey or Holm-Sidak tests as appropriate for the data. Percent incidence of AAAs was analyzed by Fishers exact test. P<0.05 values were considered to be statistically significant. Differences that attained statistical significance are represented in Tables and Figures. All data are represented as means±SEM.

Results

To determine the contribution of AT1a receptors in AngII-induced atherosclerosis and AAA formation, male LDL receptor−/− mice that were either AT1a receptor +/+ or −/− were fed a fat-enriched diet and infused with either saline or AngII (1000 ng/kg/min) for 28 days. AT1a receptor genotype had no effect on either body weight or total serum cholesterol during the study (Table 1). Systolic blood pressure was not significantly different in saline-infused AT1a receptor +/+ mice compared with −/− mice. However, AngII infusion significantly increased systolic pressure in AT1a receptor +/+ mice, while not significantly increasing AT1a receptor−/− mice (Table 1).

We determined serum titers of autoantibodies against MDA-LDL as an index of AT1a receptor deficiency on a measurement of systemically detectable oxidant stress. Neither the AT1a receptor genotype or the infusion of AngII significantly influenced these titers (supplemental Figure IA).

Recipient AT1a Receptor Genotype Is the Major Determinant of Vascular Disease in Chimeric Mice Infused with AngII

Having established the critical role of AT1a receptors in the development of AngII-induced vascular diseases, we used bone marrow transplantation to define the role of infiltrating cells expressing this receptor. To determine the efficacy of the protocol used for bone marrow transplantation in subsequent studies, we took advantage of congenic mice expressing allelic variants of the common leukocyte antigen, CD45. Irradiation leads to a rapid reduction in the number of circulating leukocytes. Subsequent to the introduction of bone marrow–derived cells, the donor cells rapidly and consistently become the predominant phenotype in the blood (supplemental Figure II). Leukocytes recovered from lavage of the peritoneal space also predominantly express the donor phenotype 8 weeks after injection (88.7±0.7% express donor CD45.1). Having demonstrated the high repopulation, the same protocol was used for all subsequent studies.

After having validated the irradiation and repopulation procedure, studies were performed in which both AT1a receptor +/+ and −/− mice were irradiated and repopulated with cells from AT1a receptor +/+ and −/− mice. All recipient and donor mice were LDL receptor–deficient. At the termination of the study, bone marrow was harvested and PCR analysis performed. These analyses demonstrated that mice had the expected genotype after irradiation and repopulation (supplemental Figure III). These 4 chimeric groups determined whether the AT1a receptors involved in AngII-induced atherosclerosis and AAAs were expressed on cells of recipient or donor origin.

The AT1a receptor genotype of the recipient or donor had no effect on body weight or serum cholesterol in groups infused with either saline or AngII (Table 2). Blood leukocytes numbers were equivalent in all groups and were unaffected by recipient or donor AT1a receptor genotype during saline or AngII infusion (Table 2). During saline infusion, there was no statistically significant difference in systolic blood pressure, irrespective of the AT1a receptor genotype of the recipient or donor. AngII infusion significantly increased blood pressure in recipient AT1a receptor +/+, but not −/− mice. The genotype of AT1a receptor on the donor cells had no significant effect on systolic blood pressure (Table 2).

The width of the suprarenal aorta was not significantly different during saline infusion, irrespective of the AT1a receptor genotype of the recipient or donor cells, and no AAAs were detected (Figure 4A and 4B). During AngII-infusion, the maximal aortic width increased significantly in both AT1a receptor +/+ recipient groups, and there was no difference based on the genotype of the donor bone marrow (Figure 4C and 4D). In contrast, in AT1a receptor−/− recipients, aortic width did not increase compared with saline, regardless of AT1a receptor donor genotype. Infusion of AngII into AT1a receptor +/+ mice repopulated with cells from +/+ mice resulted in a 58% AAA incidence. The incidence of AngII-induced AAAs was not significantly altered by repopulating AT1a receptor −/− cells into AT1a receptor +/+ recipients. Conversely, in AngII-infused AT1a receptor−/− recipients, AAAs did not develop, irrespective of the AT1a receptor genotype of the donor cells. Histological analysis revealed that aneurysmal tissue had characteristics similar to those we have described previously.5 The appearance of tissue in the suprarenal aorta of AngII-infused mice that had AT1a receptor−/− genotypes was indistinguishable from tissue obtained from saline-infused wild type mice (supplemental Figure IV).

No effect was noted of the recipient or donor genotype on serum titers of MDL-LDL autoantibodies in both saline and AngII-infused mice (supplemental Figure IB).

Discussion

In the present study, we used gene-targeted mice to demonstrate that the AT1a receptor subtype was responsible for the development of AngII-induced atherosclerosis. The dependence of AT1a receptors on the development of AngII-induced atherosclerosis is similar to previous findings demonstrating a major role for the AT1a receptor in hypercholesterolemia-induced atherosclerosis.19,27 The effect of AT1a receptor deficiency in markedly attenuating hypercholesterolemia-induced atherosclerosis has been attributed to the upregulation of the endogenous renin–angiotensin system.19 In addition, these results extend previous findings, because early reports did not demonstrate an effect of losartan administration on AngII-induced atherosclerosis.7 This lack of effect of losartan in our previous studies was probably attributable to the extensive atherosclerosis that was already present in 11-month-old apoE−/− mice. In the current study, the effect of exogenous AngII infusion was examined in LDL receptor−/− mice at 8 weeks of age that were fed a saturated fat enriched diet for 5 weeks. Thus, the effect of AT1a receptor deficiency on AngII-induced atherosclerosis was examined against a low level of preexisting atherosclerosis. Results from this and previous studies demonstrate a critical role for the AT1a receptor subtype in mediating the effects of both endogenous and exogenous AngII.

The present study also demonstrated that deletion of the AT1a receptor subtype ablated formation of AngII-induced AAAs. Previous studies in our laboratory demonstrated that pharmacological antagonism of the AT1 receptor by losartan administration totally ablated AngII-induced aneurysm formation in ApoE−/− mice.7 However, because AT1 receptor antagonists cannot discriminate between AT1a and AT1b receptor subtypes, it was unclear which receptor subtype was responsible for the effects of losartan. Both the AT1a and the AT1b receptor subtypes are expressed in the aortas of mice, with greater preponderance of the AT1b receptor particularly in the abdominal region of the aorta.28 Functional studies demonstrated that the AT1b receptor mediates the contractile response of the abdominal aorta to AngII, as contractile responses were maintained in aortic segments from AT1a receptor–deficient mice.28 Our results demonstrate a critical role for the AT1a receptor subtype in AngII-induced AAA formation. Thus, regardless of the greater preponderance of AT1b receptor expression in the abdominal aorta, it is the presence of AT1a receptors which mediates AngII-induced AAAs.

In this study, AngII-induced atherosclerosis and AAA formation were abolished in AT1a receptor–deficient mice that did not exhibit a pressor response to infusion of AngII. Thus, the lack of hypertension in AT1a receptor–deficient mice infused with AngII may have contributed to reductions in atherosclerosis or AAA formation. Previous studies demonstrated that AngII exacerbates atherosclerosis3,29 and results in AAA formation1,2,29 at infusion doses that do not appreciably elevate systolic pressure. Similarly, infusion of norepinephrine at a dose that elevated blood pressure to a similar extent as AngII did not increase atherosclerosis in ApoE−/− mice,3 or result in AAA formation.29 Recent studies demonstrate that administration of the AT1 receptor antagonist, telmisartan, to ApoE−/− mice at a dose that did not lower blood pressure suppressed atherosclerotic lesion formation.30 Collectively, these results suggest that reductions in blood pressure in AT1a receptor-deficient mice were not the primary mechanism for ablation of AngII-induced atherosclerosis and/or AAA formation in AT1a receptor–deficient mice.

Both AngII-induced atherosclerosis and AAAs are characterized by complex changes of many cell types that are both resident and infiltrates of the arterial wall. The results from whole body AT1a receptor–deficient mice demonstrate the dominant role of this protein, but do not indicate which AT1a receptor expressing cell types are interacting with AngII to generate the vascular disease. One commonly used experimental mode of discriminating the role of infiltrating cells has been use of bone marrow cell transplantation into irradiated recipients.31,32 To assist in interpretation of this experiment, we irradiated both AT1a receptor +/+ and −/− mice and repopulated with bone marrow–derived cells from these two genotypes, creating 4 groups of chimeric mice. All the donor mice were LDL receptor–deficient to circumvent the possibility of its presence influencing the development of atherosclerosis, even though this is unlikely in LDL receptor−/− recipients.33–35 If bone marrow–derived stem cells are critical to AngII-induced vascular disease, then reductions in disease with transplantation of AT1a−/− donors into +/+ recipients should be balanced by restoration of disease when +/+ donors are transplanted into −/− recipients. Using this approach, our results clearly demonstrate that AT1a receptors in the recipient are required for the initiation of AngII-induced vascular diseases, as absence of vascular pathology in AT1a receptor−/− recipients could not be overcome by AT1a receptor expression in bone marrow–derived stem cells. In AT1a receptor +/+ recipients, the repopulation with AT1a receptor−/− cells led to some attenuation of AngII-induced atherosclerosis. Thus, it appears that AT1a receptors on the recipient are required for the initiating events in the development of atherosclerosis. However, AT1a receptor expression on donor cells can exert some modulation of the development of lesion formation, but are insufficient to initiate the disease.

Although the absence of AT1a receptors on recipients had similar effects on development of both AngII-induced atherosclerosis and AAAs, this may have occurred via independent mechanisms. AngII-induced atherosclerosis is characterized by intimal accumulation of lipid-laden macrophages, in which the endothelium is a major regulator in the attraction and adherence of monocytes.2 In contrast, the earliest detectable cellular event in AngII-induced AAAs is a medial accumulation of macrophages, which may be regulated by smooth muscle cells.5 This localization may be attributable to local effects of AngII on smooth muscle cells to promote monocyte chemotaxis. Regulation of the extracellular proteolytic environment of smooth muscle cells is known to regulate AAA production.36 The development of AT1a receptor floxed mice to use in conjunction with cell-specific Cre transgenic mice will enable further refinement of the cell type(s) expressing AT1a receptors that modulate the development of these diverse AngII-induced vascular diseases.